Despite advances in the understanding of sepsis and sepsis-induced lung injury, patient morbidity and mortality remain unacceptably high. There is increasing recognition that the endothelial glycocalyx, a glycosaminoglycan-enriched endovascular layer, is a critical determinant of sepsis outcomes. The glycocalyx serves to regulate leukocyte adhesion, coagulation, microcirculatory flow, and vascular permeability ? functions vital to vascular homeostasis. Emerging preclinical and small human studies demonstrate that sepsis-mediated pathologic disturbances degrade the glycocalyx, leading to vascular dysfunction, lung injury and mortality. This increasing appreciation of the importance of glycocalyx integrity has coincided with recognition that intravenous fluid administration?long considered an essential component of sepsis resuscitation?may paradoxically worsen organ injury in sepsis. Interestingly, preclinical and small clinical studies suggest that excessive fluid resuscitation is associated with pathological glycocalyx degradation, suggesting a mechanism by which intravenous fluids may cause lung injury. Conversely, loss of glycocalyx integrity prior to sepsis resuscitation may help define a patient's susceptibility to the deleterious effects of fluids?representing a potential opportunity to personalize fluid resuscitation approaches. The Crystalloid Liberal or Vasopressors Early Resuscitation in Sepsis (CLOVERS) trial is a NIH-funded 2,320 subject multi-center, randomized, controlled clinical trial conducted by the ~50-site Prevention and Treatment of Acute Lung Injury (PETAL) network comparing alternative 24-hour fluid resuscitation strategies (liberal versus restrictive) in early sepsis and the effect on mortality. While the glycocalyx was not addressed in the original trial protocol, CLOVERS is ideally positioned to determine the potential causal associations between fluid resuscitation strategies, glycocalyx degradation, ARDS, and mortality in sepsis. The broad, long-term objectives of this proposal are to investigate if: 1) different fluid resuscitation strategies (Restrictive or Liberal) impact glycocalyx degradation in sepsis; 2) glycocalyx degradation is associated with the development of acute respiratory distress syndrome (ARDS) and/or mortality, 3) circulating markers of initial glycocalyx integrity can predict patient responses to different volume resuscitation strategies. To pursue these hypotheses, we will leverage the unique opportunity provided by the CLOVERS study to perform a comprehensive readout of glycocalyx damage, employing not only state-of-the-art measures of glycocalyx degradation (mass spectrometry detection of glycocalyx breakdown products such as heparan sulfate), but also less expensive ELISA-based (syndecan-1) and point-of-care functional assays (e.g. viscoelastic coagulation monitoring) capable of rapid assessment of glycocalyx integrity. The elucidation and improved understanding of these mechanisms may lead to strategies to predict outcomes, to select patients for tailored therapy, to follow treatment response, and to develop novel glycocalyx-directed therapies in sepsis.